This project is aimed at understanding the mechanism of folding in structural and kinetic terms for some representative model proteins. The protein folding problem remains today one of the major unsolved problems in molecular biology, despite its fundamental importance and practical implications with respect to protein engineering and biotechnology. Recent advances in two-dimensional NMR methods together with hydrogen exchange labeling and rapid mixing techniques developed in our laboratory make it possible to observe the formation of H-bonded structure at the various stages of refolding and thus to define the sequence of structural intermediates on the folding pathway. In order to elucidate the role of specific interactions in folding, these structural tools will be joined with the ability to make deliberate amino acid changes by site-directed mutagenesis. Hydrogen exchange pulse labeling and competition methods will be used to complete preliminary work on the structural description of folding intermediates in wild-type ubiquitin. This information will provide the framework for further analysis of the folding mechanism by site-directed mutagenesis. An efficient mutagenesis and bacterial expression system, based on a synthetic ubiquitin gene, is available. Mutations will be designed to alter key interactions in folding, and the resulting changes in protein stability and folding kinetics will be monitored by spectroscopic methods and 1D NMR. A limited number of mutants will be selected for more detailed pulse labeling and 2D NMR studies. Similar experimental strategies will be applied in parallel studies on wild-type and mutant forms of thioredoxin. The dominant slow folding phase of this protein will be eliminated by means of an extended pulse labeling scheme in which the protein is only briefly exposed to unfolding conditions. At a larger stage we will extend this work to ubiquitin fusion proteins in order to address the possible chaperon function of ubiquitin in ribosomal assembly and protein expression. Ubiquitin-myoglobin and ubiquitin- thioredoxin fusion proteins will be constructed and expressed in E. coli. After an initial spectroscopic characterization we will apply pulse labeling and 2D NMR methods for a more detailed study of the effect of ubiquitin attachment on the folding behavior of thioredoxin.